One of the possible ways to inhibit or disrupt the aggregation of Aβ peptide is the use of β-destroying peptides introduced by Prof. red birefringence analysis 85 4.2.1 Design of Aβ-derived BBDP containing β-switch homologous peptide 86 .
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Experimental section
Acknowledgements
Introduction
1 Introduction
Peptides and proteins
Proteins and peptides consist of a series of amino acids connected linearly via covalent bonds. Amino acids such as serine, threonine, cysteine, tyrosine, asparagine and glutamine residues fall under a category of polar residues.
Peptide bond
Amino acids are the building blocks of proteins, in which a central carbon atom called α carbon is attached to an amino group, a carboxylic acid group, a hydrogen atom and a characteristic R group. Peptide bond is planar and this is due to delocalization of electrons between the carbonyl group and the amino group as shown in Figure 2.
Torsion angles and Ramachandran plot
The Ramachandran graph is a four-quadrant graph of Psi versus Phi and tells about the permissible values of these torsion angles (Figure 5).
Secondary structures of proteins
Anti-parallel hydrogen bonding follows a simple pattern where the NH group bonds to the CO of the other strand and vice versa. However, in parallel, the NH group of the β-sheet is bonded to the CO in the adjacent strand while the CO is bonded to the NH of the amino acid residue that lies two residues away in the adjacent strand as shown in Figure 7.
Protein folding and misfolding
When a specific protein/peptide transforms from one of its functional conformations to another structure that differs from its native state, protein misfolding is referred to. This state of change in conformation leads to various diseases commonly known as protein misfolding/protein conformational diseases.
Alzheimer’s disease
- Current treatment strategies
- Aspartimide formation
- Utility of aspartimide formation
- Concept of β-breaker dipeptides in β-sheet disruption
- Biophysical methods to monitor β β β β-sheet and fibril formation
- Aim of the work
- β-breaker dipeptide containing model peptides and inhibition of self
And the in situ chemically modified peptide can be either the aspartimide-containing version of the peptide or the β-aspartyl version of the peptide. Conversion of the native α-peptide to the β-aspartyl peptide introduces flexibility in the peptide backbone and the H-bond network between the two neighboring peptides is disturbed.
Design of the β-breaker dipeptide containing model peptides
As mentioned in chapter one, for the insertion of the BBDP unit, the BBDP-containing peptide will remain as β-sheet at the initial stage, which is intended to disrupt that β-sheet architecture by aspartamide formation and subsequent ring opening at near physiological pH.
A gradient of 20% acetonitrile for 5 minutes and 60% acetonitrile for 20 minutes with a total duration of 30 minutes was used. The LC profile of the pure peptide is depicted as panel (a) in Figure 1, while the peak corresponding to the mass of the peptide is shown in panel (b).
Monitoring O to N acyl migration of peptide 1 by LC-MS
This was continued until the peaks corresponding to the aspartimide and aspartyl residues emerged. These two new peaks were assigned to the α-aspartyl and β-aspartyl residues, which were confirmed by the ESI-MS (panel d).
Monitoring stability of peptide 1 in acidic pH by LC-MS
The elution peak before the pure peptide intensified with time and we thought it to be the byproduct, benzyl alcohol. The elution peak before the peak assigned to benzyl alcohol was due to the formation of the aspartimide derivative of peptide 1, which was confirmed by the corresponding ESI-MS (panel c, figure 2).
From the deconvolution analysis, we observed 61% of the random coil conformation and 21% of the β content (Table 1). This confirms that the BBDP-containing peptide adapts the random coil conformation in PBS, which is likely due to the formation of aspartimide and aspartyl residues from the BBDP moiety.
Monitoring conformational conversion of BBDP containing peptide 1 by fourier transformation infra-red spectroscopy (FT-IR) studies
We observed the presence of a strong band at 1625 cm-1 for peptide 1 (from a 0.1% TFA-water mixture) indicating β-sheet amide I (Figure 5a). 60, 61 At the same time, a sample of the peptide from PBS was analyzed and a band at 1645 cm-1 indicating a random coil conformation was observed (Figure 5b). Thus, it is confirmed that BBDP containing peptide 1 remains in a β-sheet conformation at acidic pH, while it remains as a random coil at basic pH.
Monitoring fibril formation of peptide 1 using thioflavin T fluorescence assay
We noticed an increase in the fluorescence signal in the first 16 h, after which a decrease was noted (Figure 6). This is likely due to the fact that the model BBDP containing peptide 1 first forms β-sheet, which aggregates into fibrils.
Monitoring fibril formation of peptide 1 using transmission electron microscopy (TEM)
Those fibrils are disrupted over time, probably because the chemistry of aspartimide formation and subsequent hydrolysis occurs and peptide loses its usual peptide backbone. On the other hand, at basic pH disruption of the peptide backbone occurs via chemistry of aspartimide formation.
Monitoring amyloid formation of peptide 1 using Congo-red birefringence assay
It indicates that BBDP-containing aggregating peptide 1 forms amyloid at acidic pH, while amyloid formation is inhibited at basic pH. As a model, BBDP-containing peptide produced aggregates at acidic pH and inhibited its own aggregation in a neutral to basic pH range.
Design of model BBDP containing aggregating peptide 2
Synthesis and characterization of model BBDP containing peptide 2
The stock solution was sonicated and vortexed for 1 min each and 20 µl of the peptide sample was taken and injected into LC-MS. The mass of different forms of peptide, namely aspartimide and aspartyl residues, was observed and linked to the expected value (Fig. 10b–d).
Monitoring stability of peptide 2 in acidic pH by LC-MS
Since the BBDP -D(OBzl)S- unit was similar to that of peptide 1 , it was found that the time required for aspartimide formation and ring opening to produce aspartyl residues was also similar to that of peptide 1 .
From table 2, 54 % of the random coil conformation and 39 % of β-sheet conformation of peptide 2 were obtained. It is suggested that at that particular time of analysis conversion from β-sheet to random coil was underway.
Monitoring conformational conversion of BBDP containing peptide 2 by fourier transformation infra-Red spectroscopy (FT-IR) studies
On the other hand, peptide sample from PBS produced a band at 1640 cm-1 corresponding to random coil (Figure 13b). The disappearance of 1626 cm-1 band from the basic environment revealed that peptide no longer remains in β-sheet conformation and this is probably due to conversion of peptide from its native form to modified peptide which lacks native backbone.
Monitoring fibril formation of peptide 2 using thioflavin T fluorescence assay
We observed an increase in the fluorescence signal during the first 24 h, and then a decrease as shown in Figure 14. The reason attributed is that BBDP containing peptide 2 first aligns to form β-sheet-rich fibers and eventually fibril formation is disrupted by the chemistry of aspartimide formation, which fits our hypothesis.
Monitoring fibril formation of peptide 2 using transmission electron microscopy (TEM)
We observed the presence of a thick bundle of fibrils nearly 10 nm in diameter for peptide 2 that was incubated in a mixture of water (0.1% TFA) as shown in Figures 15a and 15b. This again confirms that the BBDP unit undergoes chemical modification in a basic environment to produce aspartimide and aspartyl residues that destroy the native backbone and consequently inhibit fibril formation.
Monitoring amyloid formation of peptide 2 using Congo-red birefringence assay
Conclusion
Inhibition of aggregation of model amyloid forming peptide using
BBDP containing model peptide 1
Introduction
- Design of model aggregating peptide
- Synthesis and characterization of peptide 3
- Kinetics of O to N acyl migration of peptide 3
- Monitoring conformational transition of peptide 3 by circular dichroism (CD) studies
- Monitoring conformational conversion of peptide 3 by Fourier Transformation Infra-Red spectroscopy (FT-IR) studies
- Monitoring fibril formation of peptide 3 using thioflavin T fluorescence assay
- Monitoring fibril formation of peptide 3 using TEM
- Monitoring amyloid formation of peptide 3 using Congo red birefringence assay
- Conclusion
No green gold birefringence was observed from sodium acetate buffer (pH 4.0) solution of the model aggregated peptide 3 after seven days of incubation time as shown in Figure 9a. This aggregates similarly to the Aβ peptide in vitro, which was demonstrated using various biophysical tools.
To investigate the β β-breaking efficiency of peptide 1 against the β β aggregation of peptide 3
- Monitoring conformational transition by circular dichroism (CD) studies
- Monitoring conformational transition by FT-IR studies
- Monitoring fibril formation using thioflavin T fluorescence assay
- Monitoring fibril formation using transmission electron microscopy (TEM)
- Monitoring amyloid formation using Congo red birefringence assay
- Conclusion
From FT-IR spectroscopic studies we observed a broad band at 1650 cm-1 for the aggregate peptide 3 when mixed with three equivalents of peptide 1 (figure 12). In parallel, 3-fold molar excess of peptide 1 (2.6 mg) was dispensed into similar eppendorf tubes (another triplicate set) containing pooling peptide 3 and kept for incubation.
Inhibition of aggregation of Aβ derived model peptide by BBDP containing
Aβ14-23 fragment itself does not form fibrils on its own.-(Leu-Ser)n- residues probably help to promote the aggregation and solubility of the peptide sequence, similar to model aggregating peptide. The effectiveness of our relevant β-breaker peptide wanted to be tested on the aggregation of this peptide.
Synthesis and characterization of peptide 4
More importantly, one serine switch element is included which controls the self-assembly and which can be regulated by the pH of the system. Glycine was used as a spacer at C-terminus, as it helps to attach to the resin.
Monitoring O to N acyl migration of peptide 4
At zero time we noted one major peak at 3.85 min, corresponding to the peptide in the depsi form. Our next goal was to investigate the β-sheet-forming potential of Aβ-derived aggregating peptide 4 in a specific state using various biophysical tools.
Monitoring conformational conversion of peptide 4 by circular dichroism (CD) studies
We expect that at acidic pH no acyl displacement occurs as a result, the peptide exists in random coil conformation and vice versa. We observed an equal ratio of random coiling to β-sheet for peptide 4 from sodium acetate buffer and this may be due to the high concentration of the peptide which is forced to aggregate even at acidic pH.
Monitoring conformational conversion of peptide 4 by FT-IR studies
Monitoring fibril formation of peptide 4 using thioflavin T fluorescence assay
So this confirms that in basic pH peptide 4 aggregates to form fibrils which bind to thioflavin T. This is probably because under basic condition O to N acyl migration takes place, the peptide regains its original backbone, it forms β-sheet and aggregates to form fibrils.
Monitoring fibril formation of peptide 4 using TEM
On the other hand, we observed long needle-like fibrils of nearly 10 nm in diameter for the peptide 4 incubated in PBS. This TEM investigation confirms that peptide 4 forms fibrils in basic pH and can be used as a substitute for Aβ peptide.
Monitoring amyloid formation of peptide 4 using Congo-red birefringence assay
No aggregates were observed in the sample incubated in sodium acetate buffer, pH 4.0 (Fig. 6a-b). From the above experimental findings, it was clear that Aβ-derived aggregating peptide 4 aggregates in a controlled manner and also forms amyloid-like fibrils that can be used as a mimic of full-length Aβ for testing our concept of β-sheet disruption .
With a mimic in hand, our next goal was to design and synthesize suitable breaker peptide.
Synthesis and characterization of peptide 5
Monitoring aspartimide formation of peptide 5 by LC-MS
After monitoring chemical conversion of BBDP unit of peptide 5 to aspartimide derivative and further to aspartyl residues using LC-MS, our next aim was to investigate β-sheet-forming potential and inhibition of self-assembly of peptide 5 at a specific pH.
Monitoring conformational conversion of peptide 5 by circular dichroism (CD) studies
The CD spectrum of peptide 5 from sodium acetate buffer showed a characteristic minimum at 220-225 nm and a maximum at 195-200 nm, a typical β-sheet signal. From the experiment, it was found that peptide 5 aggregates in sodium acetate buffer to form fibrils with a β-sheet-rich architecture.
Monitoring conformational conversion of peptide 5 by FT-IR
This is due to the chemistry of aspartimide formation that occurred at basic pH and peptide 5 remained in random coil conformation after three days. This indicates an inhibition of β-sheet formation by peptide 5 at basic pH after three days.
Monitoring fibril formation of peptide 5 using thioflavin T fluorescence assay
We observed a slow increase in fluorescence in the initial stages, which continued for three days, as shown in Figure 13. This may be due to the fact that after three days, fibril formation was inhibited due to the chemistry of aspartimide formation and subsequent ring opening.
Monitoring fibril formation of peptide 5 by TEM
Experiment with TEM confirmed the fibril formation of peptide 5 in an acidic condition where native backbone is retained. Once the pH was triggered to basic condition due to aspartimide formation, the backbone is disrupted and due to which fibril formation is inhibited.
Monitoring amyloid formation of peptide 5 using Congo-red birefringence assay
On the other hand, at basic pH, fibril and amyloid formation were not noted in TEM and birefringence experiments, respectively.
To investigate the β β β-breaking efficiency of peptide 5 against the β aggregation of peptide 4
- Monitoring conformational transition by CD studies
- Monitoring conformational transition using FT-IR
- Monitoring fibril formation using thioflavin T fluorescence assay
- Monitoring fibril formation using transmission electron microscopy (TEM)
- Monitoring fibril formation using Congo red birefringence assay
- Conclusion
This indicates that three equivalents of peptide 5 could inhibit β-sheet formation of peptide 4 at pH 7.4. Three-day-old stock solution was used to analyze the conformation of peptide 4 when mixed with peptide 5 in three equivalents.
- Synthesis and characterization of β-breaker peptide 6 and 7
- Kinetics of aspartimide formation
- Monitoring conformational changes by CD
- Monitoring conformational changes by FT-IR
- Monitoring fibril formation using thioflavin T fluorescence
- Monitoring fibril formation by electron microscopy (TEM)
- Monitoring amyloid formation using Congo red birefringence assay
- Conclusion
- Modulation of aggregation propensity of Aβ ββ β38 by site specific multiple
The fluorescence was measured at an excitation wavelength of 435 nm and an emission wavelength at 485 nm (final concentration of peptide 2.5 µM and ThT is 25 µM). Congo red photographs of the peptide sample (a) Optically polarizable microscope image of the A when mixed with ten equivalents of Ac-LD(OBzl)FFD-NH2. .. formation using Congo red birefringence test.
- Design of soluble version of Aβ β β β38
- Synthesis and Characterization of Aβ β β β38
- Synthesis and Characterization of P3-Aβ β β38 β
- Monitoring the secondary structure of Aβ β β β38 and P3-Aβ β β β38 by circular dichroism (CD) studies
- Monitoring the conformation of Aβ β β β38 and P3-Aβ β β β38 by FT-IR
- Monitoring fibril formation of Aβ β β β38 and P3-Aβ β β β38 by thioflavin T fluorescence
- Monitoring fibril formation of Aβ β β β38 and P3-Aβ β β38 by TEM β
- Monitoring amyloid formation by Aβ β β38 and P3-Aβ β β β β38 using Congo red birefringence study
- Conclusion
- Experimental section
- Instrumentation and general methods
- Solid Phase Peptide Synthesis
On the other hand, the secondary structure of P3-Aβ38 was characterized by a minimum centered at 198 nm (black curve, figure 3). Aβ38 and P3-Aβ38 samples were prepared at the same time and examined using electron microscopy.
![Figure 1: (a) HPLC profile picture of peptide and observed mass 1377.67, [M+4H] 4+](https://thumb-ap.123doks.com/thumbv2/azpdfnet/10495242.0/139.892.168.733.288.932/figure-hplc-profile-picture-peptide-observed-mass-1377.webp)
Synthesis
Synthesis of the model that aggregates the peptide H2N-Ser-Leu-Ser-Leu-(H+)Ser-Leu-Ser-Gly-NH2 (3). After the final deprotection of the last Fmoc group, the peptide was cleaved from the resin using TFA/DCM (4:1) for 3 h and precipitated using cold diethyl ether.
List of Publications
Eur waste reduction in amide synthesis by a continuous method based on recycling the reaction mixture" RSC amide synthesis by a continuous method based on recycling the reaction mixture" RSC Adv.
